Effect of phosphodiesterase (1B, 2A, 9A and 10A) inhibitors on central nervous system cyclic nucleotide levels in rats and mice.

Phosphodiesterase (PDE) inhibition has been broadly investigated as a target for a wide variety of indications including central nervous system (CNS) disorders. Cyclic nucleotide (cNT) changes within associated tissues may serve as a biomarker of PDE inhibition. We recently developed robust sample harvesting and bioanalytical methods to quantify cNT levels in rodent brain and cerebrospinal fluid (CSF). Herein, we report on the application of those methods to study rodent species-specific and rodent brain region-specific cNT changes following individual or concomitant PDE inhibitor administration. Male Sprague Dawley (Crl:CD® [SD]) rats were dosed subcutaneously (sc) with a PDE1B inhibitor (DNS-0056), a PDE2A inhibitor (PF-05180999), a PDE9A inhibitor (PF-4447943), and a PDE10A inhibitor (MP10), each at a single dose of 10 or 30 mg/kg, or concomitantly with all 4 inhibitors at 10 mg/kg each. Male Carworth Farms (Crl:CF1 ®[CF-1]) mice were dosed intraperitoneally (ip) with the four individual inhibitors at a single dose of 10 mg/kg or concomitantly with all 4 inhibitors at 10 mg/kg each. The doses studied are generally adequate for affecting measurable cNT levels in the tissues of interest and were thereby chosen for this investigation. Measured 3',5'-cyclic adenosine monophosphate (cAMP) changes were generally statistically insignificant in the brain, striatum and CSF after administration of the aforementioned PDE inhibitors. However, the levels of 3',5'-cyclic guanosine monophosphate (cGMP) increased in both rat and mouse striatum (2.2-, 2.1- and 1.7-fold and 6.4-, 2.8- and 1.7-fold, respectively) after PDE2A, 9A, and 10A inhibitor dosing. In all cases, the cNT changes followed the same trend in the brain, striatum and CSF after PDE inhibitor dosing and dose response was observed in rats. Concomitant treatment with PDE1B, PDE2A, PDE9A and PDE10A inhibitors resulted in a 4.4- and 36.7-fold increase of cGMP in rat and mouse striatum. The drug exposures after concomitant treatment were also higher than in the individual inhibitor-treated animals. cGMP enhancement observed could be due to synergistic effects, though an additive effect of the combined inhibitor concentrations may also contribute.

Design and Synthesis of Novel and Selective Glycine Transporter-1 (GlyT1) Inhibitors with Memory Enhancing Properties.

We report here the identification and optimization of a novel series of potent GlyT1 inhibitors. A ligand design campaign that utilized known GlyT1 inhibitors as starting points led to the identification of a novel series of pyrrolo[3,4- c]pyrazoles amides (21-50) with good in vitro potency. Subsequent optimization of physicochemical and in vitro ADME properties produced several compounds with promising pharmacokinetic profiles. In vivo inhibition of GlyT1 was demonstrated for select compounds within this series by measuring the elevation of glycine in the cerebrospinal fluid (CSF) of rats after a single oral dose of 10 mg/kg. Ultimately, an optimized lead, compound 46, demonstrated in vivo efficacy in a rat novel object recognition (NOR) assay after oral dosing at 0.1, 1, and 3 mg/kg.

Linear Epitopes in Vaccinia Virus A27 Are Targets of Protective Antibodies Induced by Vaccination against Smallpox.

UNLABELLED: Vaccinia virus (VACV) A27 is a target for viral neutralization and part of the Dryvax smallpox vaccine. A27 is one of the three glycosaminoglycan (GAG) adhesion molecules and binds to heparan sulfate. To understand the function of anti-A27 antibodies, especially their protective capacity and their interaction with A27, we generated and subsequently characterized 7 murine monoclonal antibodies (MAbs), which fell into 4 distinct epitope groups (groups I to IV). The MAbs in three groups (groups I, III, and IV) bound to linear peptides, while the MAbs in group II bound only to VACV lysate and recombinant A27, suggesting that they recognized a conformational and discontinuous epitope. Only group I antibodies neutralized the mature virion in a complement-dependent manner and protected against VACV challenge, while a group II MAb partially protected against VACV challenge but did not neutralize the mature virion. The epitope for group I MAbs was mapped to a region adjacent to the GAG binding site, a finding which suggests that group I MAbs could potentially interfere with the cellular adhesion of A27. We further determined the crystal structure of the neutralizing group I MAb 1G6, as well as the nonneutralizing group IV MAb 8E3, bound to the corresponding linear epitope-containing peptides. Both the light and the heavy chains of the antibodies are important in binding to their antigens. For both antibodies, the L1 loop seems to dominate the overall polar interactions with the antigen, while for MAb 8E3, the light chain generally appears to make more contacts with the antigen. IMPORTANCE: Vaccinia virus is a powerful model to study antibody responses upon vaccination, since its use as the smallpox vaccine led to the eradication of one of the world's greatest killers. The immunodominant antigens that elicit the protective antibodies are known, yet for many of these antigens, little information about their precise interaction with antibodies is available. In an attempt to better understand the interplay between the antibodies and their antigens, we generated and functionally characterized a panel of anti-A27 antibodies and studied their interaction with the epitope using X-ray crystallography. We identified one protective antibody that binds adjacent to the heparan sulfate binding site of A27, likely affecting ligand binding. Analysis of the antibody-antigen interaction supports a model in which antibodies that can interfere with the functional activity of the antigen are more likely to confer protection than those that bind at the extremities of the antigen.

Protection of rabbits and immunodeficient mice against lethal poxvirus infections by human monoclonal antibodies.

Smallpox (variola virus) is a bioweapon concern. Monkeypox is a growing zoonotic poxvirus threat. These problems have resulted in extensive efforts to develop potential therapeutics that can prevent or treat potentially lethal poxvirus infections in humans. Monoclonal antibodies (mAbs) against smallpox are a conservative approach to this problem, as the licensed human smallpox vaccine (vaccinia virus, VACV) primarily works on the basis of protective antibody responses against smallpox. Fully human mAbs (hmAbs) against vaccinia H3 (H3L) and B5 (B5R), targeting both the mature virion (MV) and extracellular enveloped virion (EV) forms, have been developed as potential therapeutics for use in humans. Post-exposure prophylaxis was assessed in both murine and rabbit animal models. Therapeutic efficacy of the mAbs was assessed in three good laboratory practices (GLP) studies examining severe combined immunodeficiency mice (SCID) given a lethal VACV infection. Pre-exposure combination hmAb therapy provided significantly better protection against disease and death than either single hmAb or vaccinia immune globulin (VIG). Post-exposure combination mAb therapy provided significant protection against disease and death, and appeared to fully cure the VACV infection in ≥50% of SCID mice. Therapeutic efficacy was then assessed in two rabbit studies examining post-exposure hmAb prophylaxis against rabbitpox (RPXV). In the first study, rabbits were infected with RPVX and then provided hmAbs at 48 hrs post-infection, or 1 hr and 72 hrs post-infection. Rabbits in both groups receiving hmAbs were 100% protected from death. In the second rabbitpox study, 100% of animal treated with combination hmAb therapy and 100% of animals treated with anti-B5 hmAb were protected. These findings suggest that combination hmAb treatment may be effective at controlling smallpox disease in immunocompetent or immunodeficient humans.

Combination therapy of vaccinia virus infection with human anti-H3 and anti-B5 monoclonal antibodies in a small animal model.

BACKGROUND: Treatment of rare severe side effects of vaccinia virus (VACV) immunization in humans is currently very challenging. VACV possesses two immunologically distinct virion forms in vivo - intracellular mature virion (MV, IMV) and extracellular virion (EV, EEV). METHODS: Antibody-mediated therapeutic efficacy was determined against VACV infection in a small animal model of progressive vaccinia. The model consisted of severe combined immunodeficiency mice infected with VACV New York City Board of Health vaccine strain and treated with monoclonal antibodies (mAbs). RESULTS: Here, we show that combination therapy with two fully human mAbs against an immunodominant MV antigen, H3 (H3L), and an EV antigen, B5 (B5R), provides significantly better protection against disease and death than either single human monoclonal or human vaccinia immune globulin, the currently licensed therapeutic for side effects of smallpox vaccination. CONCLUSIONS: The preclinical studies validate that this combination of mAbs against H3 and B5 is a promising approach as a poxvirus infection treatment for use in humans.